Molecular Diagnosis
The molecular diagnosis of diseases and genetic traits is an emerging field, particularly in the area of clinical analysis. Generally, it uses techniques from molecular biology for the study of DNA/RNA, of infectious agents, or of genetic changes in the organism itself, aiding in the diagnosis and prognosis of infectious and genetic diseases.
The more common molecular biology techniques currently used are: enzymatic amplification of the DNA (PCR), digestion of the genomic DNA strand or of PCR product with restriction enzymes, electrophoretic separation of the DNA or of the PCR product, hybridization of the DNA or PCR fragments with oligonucleotide probes, DHPLC and cytogenetic methods. These techniques allow the rapid genotyping of polymorphic markers, tracking of uncharacterized mutations. In particular, the cytogenetic methods, based on the microscopic observation of normal and abnormal chromosomes, allow the construction of cytogenetic maps of the genomes of many species. The FISH (fluorescent in situ hybridization) cytogenetic method is the most direct means of locating molecular and genetic markers in the cytogenetic map, allowing the integration between genetic and molecular markers. Probes are widely used for diagnosis, such as cosmid probes, which are unique sequences connected in small segments of certain chromosomes, being useful for the study of microdeletions. Other probes are used to detect translocations and highly repetitive sequences. However, one should point out that some of these techniques still have some limitations, such as false-positive signals, that can lead to an error in diagnosis.
A well-known molecular diagnosis system is the ELISA (Enzyme-Linked ImmunoSorbent Assay). This immune-enzymatic test allows the detection of specific antibodies in the serum of patients, being the first-line test in the diagnosis of HIV (human immunodeficiency virus) infection. The method for performing the test is based on the antibody-antigen interaction, with this test also being capable of detecting other substances, such as hormones.
The present invention refers to the fluorescent nanoparticle composites themselves, method for the preparation of these composites, system for rapid diagnosis (as “kits”) containing such compounds, and functioning of said “kits”. In particular, the composites of the present invention have specific characteristics regarding size and fluorescence, and have an affinity for biological molecules, such as DNA, RNA, and also proteins. The method for the preparation of these compounds is also described in the present invention. Plus, the present invention describes the method of preparation for an adequate probe (named here as “support”) containing biological material of the organism one wishes to study. Upon this support the fluorescent nanoparticle composites and the patient's biological material are added, comprising a diagnostic system, designated here as the ELINOR (from “Enhanced Luminescence from Inorganic/Organic nanocomposites”) test, for the diagnosis of diseases caused by several pathogens and/or genetic diseases, amongst other things. The present invention has application mainly in the medical and veterinarian fields.
The patent literature describes an ample variety of probes for the diagnosis of specific diseases. However, most of the documents deal with methods that use the PCR molecular biology technique, requiring the amplification of the biological molecule that one wishes to study in order to perform the diagnosis. One can exemplify the methods for the diagnosis of diseases by the documents presented below.
Document U.S. Pat. No. 6,258,570 deals with a method for the diagnosis of viral meningitis using PCR, as does document U.S. Pat. No. 7,041,255, which uses the same technique to detect infection by the dengue virus. Likewise, the PCR is used for the diagnosis of the human papilloma virus (HPV), as described by document U.S. Pat. No. 6,027,89, and of Streptococcus pneumoniae, as described by U.S. Pat. No. 6,869,767.
The present invention differs from all of those documents by not requiring a step of amplification (such as the one performed in the PCR technique) in order to perform the molecular diagnosis.
The patent literature also reveals several examples of fluorescent biosensors containing gold, out of which we highlight the most relevant.
Document US 2007/0059693 describes a biosensor containing a fluorescent surface, molecules of nucleic acid, and a fluophore. The fluorescent surface may be a metal, including gold. The molecules of nucleic acid must have one of the ends bound to the fluorescent surface and the other end to a fluophore. This molecule of nucleic acid may also have internal hybridization regions that, when hybridized, form a “staple”. In these cases, the fluophore will be close to the fluorescent surface, allowing fluorescence to occur. The present invention differs from that document due to the support surface not being necessarily fluorescent nor metallic, and not requiring that the molecules of nucleic acid form a “staple” in order to emit fluorescence.
Document US 2005/0196876 describes a method for the analysis of the content of a biological sample through the contact of the sample with a nanoporous biosensor. This biosensor contains probes that bind to the samples forming complexes that will be bound to a second probe. That probe will be illuminated so as to send a specific fluorescent signal. In an optional configuration, this biosensor may have a layer of gold. The present invention differs from the aforementioned document by dealing with fluorescent nanoparticles containing gold, there being no need to bind to more than one probe.
Document U.S. Pat. No. 6,773,884 describes a method for the detection of nucleic acids in which those molecules are put in contact with one or more nanoparticles of gold bound to oligonucleotides and to fluorescent molecules. When the hybridization occurs, the interaction of these molecules with the oligonucleotides suffers an alteration detectable as changes in the florescence. The present invention differs from the aforementioned document by dealing with nanoparticles in which the gold is covered by polymers, and by being deposited over the biological molecules studied, there being no need for the presence of oligonucleotides bound to the nanoparticle.
Document U.S. Pat. No. 7,083,928 describes the detection of negatively charged polymers using water-soluble cationic polythiophenes. The negatively charged polymers include biological molecules such as nucleic acid. This polymer may be bound to a conductive support, such as a gold surface. When the polymer is detected, there is a change in the electronic load, fluorescence, or color, The present invention differs from the aforementioned document by dealing with nanoparticles of gold covered by polymers that interact with the biological molecules, with the gold not being part of the adequate support that will immobilize the biological molecules.
Therefore, no document was found describing, nor suggesting, the fluorescent nanoparticle composites themselves, their form of preparation, the systems containing such composites for use in diagnostic “kits”, or form of functioning for such systems.